Quiescin sulfhydryl oxidases (QSOX) are flavin-dependent enzymes that catalyze the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. They were originally associated with quiescence in fibroblasts, and were later shown to have sulfhydryl oxidase activity. The QSOX family of enzymes is formed from the fusion of thioredoxin domains and FAD-binding domains (ERV1/ALR). QSOX genes have been identified in all multicellular plants and animals and in some protista, but have not been found in fungi. (Reviewed in D. Coppock and C. Thorpe, Antioxidants & Redox Signalling 8: 300-311, 2006; C. Thorpe and D. Coppock, J. Biol. Chem. 282: 13929-13933, 2007).
The QSOX enzymes are distinct from fungal sulfhydryl oxidases, which are actually glutathione oxidases (H. Kusakabe, et al., Agric. Biol. Chem. 46: 2057-2067, 1982; K. Hoober et al., J. Biol. Chem. 274: 31759-31761, 1999) and the metal-dependent sulfhydryl oxidase reported by V. G. Janolino and H. E. Swaisgood, J. Biol. Chem. 250: 2532-2538, 1975; Food Science and Technology 122: 539-546, 2003. QSOX enzymes exhibit much greater activity in protein-cysteine oxidation than fungal glutathione oxidases (K. Hoober et al., J. Biol. Chem. 274: 22147-22150, 1999; C. Thorpe, et al., Arch. Biochem. Biophys. 405: 1-12, 2002).
Kusakabe et al. have shown that the sulfhydryl oxidase from Penicillium sp. has undetectable activity in introducing disulfides into protein substrates, using standard assays employing RNAse as a standard protein for oxidative protein folding studies. H. Kusakabe, et al., Agric. Biol. Chem. 46: 2057-2067, 1982.
Similarly, de la Motte and Wagner, as well as Janolino and Swaisgood, using RNAse as a substrate, have shown that the Aspergillus sulfhydryl oxidase has extremely low activity (less than 0.02 thiols oxidized per minute). R. S. de la Motte and F. W. Wagner, Biochemistry 26: 7363-7371, 1987; V. G. Janolino and H. E. Swaisgood, Milchwissenschaft 47: 143-146, 1992.
A comparison of the distinctive properties of QSOX1 (quiescin Q6 sulfhydryl oxidase 1) and metal-dependent sulfhydryl oxidase from milk (MetalSox) is shown in Table 1.
TABLE 1Comparison of the properties of purified QSOX1 from milkand reported metal-dependent sulfhydryl oxidase from milkPropertyMilk-QSOX1MetalSOXMolecular weight62 kDa89 kDaCofactor contentFlavin (FAD)Iron (Fe)Is activity stimulatedNoYesby added Ferrous iron?Is EDTA inhibitory?NoYesSequence informationYesNoAvailable?Catalytic efficiency3.74 × 105Not availablewith reduced RNasein the literature(kcat/Km (M−1s−1)) perthiol
A QSOX enzyme isolated from human placental tissue has also been shown to have activity in stimulating cell proliferation and growth in normal and cancer cells, as described in U.S. Pat. No. 6,075,008 (SEQ ID NOs: 4 and 6), and may have therapeutic uses.
Avian QSOX1 can be isolated from egg white, which yields approximately 0.4 mg QSOX1 per 100 g of egg white protein (Hoober et al., J. Biol. Chem. 271, 30510-30516, 1996). However, the high viscosity of egg white makes isolation and purification of QSOX1 from this source very difficult. Human QSOX1 has been localized in a number of human tissues by immunohistochemistry, but is present only in very small amounts in these tissues (Coppock and Thorpe, Antioxidants & Redox Signalling 8: 300-311, 2006).
Whey is a liquid by-product from the preparation of cheese or casein from cow's milk. For every pound of cheese produced, approximately 9 pounds of liquid whey result. It has been estimated that dairies in the state of Wisconsin alone produce about 19 billion pounds of liquid whey byproduct annually from cheese production, which incurs substantial costs in disposal and waste management. (K. Parmentier, Determination of the Volume of Industrial Waste from Wisconsin's Dairy Products Industry, www.wastenot-organics.wisc.edu/library/whey). In 1996, it was reported that worldwide, volumes of whey accumulated at greater than 80×109 liters per year (G. W. Smithers, et al., J. Dairy Sci. 79: 1454-1459, 1996). Whey has some limited commercial uses as an additive in processed foods and animal feed, as a nutritional supplement, and as a commercial source of lactoferrin and lactoperoxidase. (G. W. Smithers, et al., J. Dairy Sci. 79: 1454-1459, 1996; M. R. Etzel, J. Nutrition. 134, 996S-1002S, 2004).
Quiescin sulfhydryl oxidases (QSOX) are useful in generating disulfide bonds in small molecules, and in unfolded, reduced peptides and proteins, thereby facilitating protein folding, and in generating intermolecular and intramolecular disulfide bonds, for example, in the formation of disulfide-bridged networks and gels. QSOX enzymes use only oxygen as an oxidant and are the most versatile disulfide bond-forming enzymes yet known. In addition, they may also be used as enzymatic oxidants for sulfhydryl compounds in aqueous solution in the presence of molecular oxygen, and as an alternative to chemical oxidants in the re-oxidation of hair and wool fibers, or of other materials reduced with suitable reductants. Although other sulfhydryl oxidases have been reported, only QSOX enzymes have been shown to readily insert disulfide bonds into a wide range of sulfhydryl compounds, in particular, peptides and proteins. (K. L. Hoober et al., J. Biol. Chem. 274: 22147-22150, 1999; C. Thorpe and D. L. Coppock, J. Biol. Chem. 282: 13923-13933, 2007). Due to the superior performance of QSOX enzymes in creating disulfide bonds and mediating oxidative protein folding, a need exists for a cost-effective method to provide these enzymes commercially.
Despite these important industrial and medical uses, there is currently no known purification procedure of any QSOX enzyme that could be adapted for industrial scale purification of QSOX proteins, in part because no feasible source of QSOX for industrial purification has been found.